ABSTRACT Protein phosphorylation by kinases with its converse dephosphorylation by phosphatases regulates most biological processes. In human cells, more than three-quarters of cellular proteins are phosphorylated. The occupancy of a given phosphorylation site reflects the balance between the activities of kinases and phosphatases. To understand the reversible nature of protein phosphorylation, we must investigate the forward and reverse reaction by connecting kinases and phosphatases on their shared substrates of interest. Protein phosphorylation is catalyzed by more than 500 protein kinases, however, most protein dephosphorylation is carried out by only seven phosphoprotein phosphatases (PPPs). While there has been tremendous progress in deciphering cellular signaling by kinases, much less is known about phosphatases. Research in my laboratory is focused on integrating phosphatases into cellular signaling networks by establishing phosphatase-substrate relationships, identifying opposing kinases, and determining regulatory inputs. My laboratory is uniquely positioned to address these outstanding challenges by combining quantitative proteomics and phosphoproteomics approaches in cells with reconstitution of minimal signaling units in vitro. PPP form multimeric holoenzymes with overlapping subunits that function as distinct entities, which hampers mechanistic studies of holoenzyme specific functions and regulation in cells. My research program directly addresses this problem by studying PPP signaling networks on a system-wide level in cells and with isolated components in vitro. By combining in-cell discovery and in vitro validation and mechanistic reduction, we will distinguish direct from indirect effects or cellular compensation mechanisms and determine the contribution of specific holoenzymes. In this application we will focus on PP6, a PPP with limited subunit diversity that nonetheless accurately reflects the combinatorial nature of PPP holoenzymes. Collectively, these findings will further our understanding of holoenzyme specific phosphatase function, substrate preferences, and regulation, and connect phosphatase and kinase biology by integrating them into functional networks. Defining reciprocal PPP ? protein kinase regulation and opposition of shared substrates will provide insights into how physiological processes are controlled by reversible phosphorylation. We will continue to develop and implement innovative approaches in proteomics and cell biology to better address these and emerging questions. We envision this work to be a resource for the phosphorylation signaling community, as well as a framework for future research into phosphatase biology. We will share our data, reagents, and experimental approaches and generate an easily accessible database for the phosphatase substrates we will identify.